Testkit and method

ABSTRACT

Skin prick test for the determination of the predisposition of an individual to develop marginal periodontitis, said kit comprising: (a) a first reagent containing a known quantity of a surface structure common to anaerobic Gram negative pathogens which is capable of triggering the inflammatory response associated with periodontitis and gingivitis; (b) second reagent containing an agonist to said individual; (c) a negative control; and (d) instructions for the use of said kit; and a method for such determination of predisposition.

The present invention relates to a skin prick test kit for thedetermination of the predisposition of an individual to develop marginalperiodontitis. The invention also involves a method for thedetermination of such predisposition.

BACKGROUND OF THE INVENTION

The most frequent disease that affects the attachment apparatus of theteeth or the periodontium is gingivitis. It is caused by our indigenousbacterial flora and is, for some individuals a first step towardsmarginal periodontitis. A shift towards more anaerobic bacteria in thebacterial plaque is thought to be the reason why gingivitis turns intomarginal periodontitis. Despite the fact that almost all individualssuffer from gingivitis, there is no clear relation between the degree ofgingivitis and development of marginal periodontitis. Epidemiologicalstudies have, however, shown that between 5 and 10% of the adultpopulation develop severe marginal periodontitis, while a moderate formaffects 50 to 80%.

Progression of periodontitis is influenced by a multitude of modifyingfactors (Genco, RJ, Löe H. The role of systemic conditions and disordersin periodontal disease. Periodontol 2000 1994; 2: 98-116). These can bedivided into systemic factors which affect the entire dentition andlocal factors which affect a single tooth or tooth surface.

The teeth are attached to the alveolar bone through their roots. A thinlayer of mineralized cementum is found along the surface of the roots.The cementum layer anchors collagen fibers which extend to the adjacentalveolar bone. The space, thus created between the root and the bonesurfaces, is occupied mainly by collagen fibers and connective tissuecells (fibroblasts). The soft tissue, known as the periodontal membraneor ligament, is a highly specialized connective tissue. It has thecapacity to form bone as well as cementum and can, provided the rightconditions are given, form a new attachment apparatus in areas of theroot where it has been lost to periodontal disease (Lindskog S,Lengheden A, Blomlöf L. Successive removal of periodontal tissues.Marginal healing without plaque control. J. Clin. Periodontol 1993; 20:14-9).

Periodontal disease is, second to tooth decay, the most frequent oraldisease. It is a progressive disease and affects, in its severe form, 5to 10% of the population in industrialized countries, leading to partialor complete tooth loss. However, most adults have one or more teethaffected by the disease. The disease is, in its most common form knownas marginal periodontitis. It is caused by accumulation of bacterialdeposits on tooth surfaces along the gingival margins. These bacteria,predominantly anaerobic originate from the hosts indigenous oralmicroflora and elicit an inflammatory reaction in the gingiva whichresults in destruction of tooth-supporting tissues (periodontal membraneand alveolar bone). The destruction of tooth-supporting tissues resultsin a deepening of the space (periodontal pocket) between the root of thetooth and the gingiva. The disease progresses as bacteria migrateapically into the periodontal pocket, which deepens more and more as aresult of the soft tissue inflammation. Unless adequate treatment isinstigated, the tooth becomes mobile and will eventually fall out whenenough tooth-supporting tissues have been destroyed.

Three theories on or models for progression of marginal periodontitishave been proposed (Socransky SS, Haffajee AD, Goodson J, Lindhe J. Newconcepts of destructive periodontal disease. J. Clin Periodontol 1984;11: 21-32):

1. A slow continuous loss of attachment throughout life.

2. Random periods of attachment loss throughout life.

3. Localized attachment loss during certain periods in life.

There is reason to believe that all three theories are valid, eachwithin a certain subpopulation of patients suffering from marginalperiodontitis. The first theory/model applies to the majority ofpatients, 50 to 80% of the population, which show a moderate form ofmarginal periodontitis. The third theory/model applies to juvenileperiodontitis which affects young patients and then most often only thefirst permanent molars. The second theory/model accounts for the 5 to10% of the population which suffer from severe marginal periodontitis.

Within the group of patients suffering from severe marginalperiodontitis and which lose periodontal attachment during randomperiods throughout life (theory/model 2 above) a large number ofpatients fail to respond to treatment (refractory patients). Thesepatients have most probably a “multi-factorial genetic predisposition”to develop severe marginal periodontitis.

This genetic predisposition has been suggested to lead to aninflammatory overreaction to marginal bacterial plaque which in turncauses destruction of the marginal periodontium.

SUMMARY OF THE INVENTION

The main object of the present invention is to provide new techniquesresiding in a screening test to identify within a given populationindividuals who have predisposition to develop marginal periodontitis.

Another object of the present invention is to provide a skin prick testkit for such determination of predisposed individuals.

Another object of the invention is to provide a method for thedetermination of such predisposition of individuals within a givenpopulation.

For these and other objects which will be clear from the followingdisclosure the invention provides for a skin prick test kit for thedetermination of the predisposition of an individual to develop marginalperiodontitis, said kit comprising:

a) a first reagent containing a known quantity of a surface structurecommon to anaerobic Gram negative pathogens which is capable oftriggering the inflammatory response associated with periodontitis andgingivitis;

b) a second reagent containing an agonist to said individual;

c) a negative control; and

d) instructions for the use of said kit.

It is preferred that said first reagent contains a structure selectedfrom the non-polysaccharide entity of surface lipo- or proteoconjugatesof such anaerobic Gram negative pathogens.

It is particularly preferred that said conjugate is alipopolysaccharide, and in such conjugate it is particularly preferredthat said entity is Lipid A or an equivalent thereof of same biologicalfunction.

In regard to the second reagent said agonist can be any substancecapable of producing an inflammatory response on challenge of anindividual subject to test. Although any agonist of such function can beused it is preferably selected from histamine, serotonin,prostaglandins, leukotrienes and bradykinin.

It is particularly preferred to use as an agonist histamine which is aconventional challenge substance used in testing immunologic reaction.

The invention also provides for a method for the determination of thepredisposition of an individual to develop marginal periodontitis. Insuch a method the skin prick test is performed by challenging anindividual subject to testing with a first reagent as defined above,evaluating the reaction of said challenge. In this method lack ofreaction or generation of only a minor reaction indicates predispositionto develop a marginal periodontitis.

As will be shown below by experimental data patients refractory totreatment surprisingly present a reverse response, i.e. they do notrespond as readily as healthy individuals with an inflammatory reactionto a given common Gram negative bacterial antigen, such as Lipid A. Thisimpaired response could therefore lead to an unimpaired or uncontrolledgrowth of the marginal microflora, especially the anaerobic subgingivalpathogens which, when they reach a critical volume, initiate a period ofmarginal attachment loss. The techniques presented by the presentinvention residing in the administration of a suitable antigen, such asLipid A, thus has the potential of serving as a screening test for theearly identification of individuals which have a predisposition todevelop severe marginal periodontitis.

In regard to the first reagent any antigen or surface structure commonto Gram negative bacterial periodontitis pathogens can be used in thetest method according to the present invention. Such antigens arefrequently part of surface conjugates of said pathogens, such as lipo-or proteopolysaccharides. Since the polysaccharide part of suchconjugates has a structure varying with the type of bacterial straininvolved, it is preferred to use the non-polysaccharide entity of suchconjugates, said entity being unaltered regardless of bacterial strain.

With regard to the second reagent used in the present invention toconfirm a positive reaction, it goes without saying that any agonist canbe used for such purpose. Furthermore, the negative control forconfirming negative reaction can be simply constituted by pyrogene freewater.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further illustrated by specificexperimental data, but it is to be noted that the invention is notrestricted to the experiments presented below since alterations andmodifications will be obvious to the skilled artisan upon reading of thedisclosure.

EXAMPLE Skin Prick Test Using Lipid A

Lipid A is a unique part of endotoxins, lipopolysaccharides (LPS)produced by all periodontitis pathogens. Unlike the polysaccharide partof LPS, it remains unaltered regardless of bacterial strain. LPStriggers a number of biological activities most notably the inflammatoryresponse in periodontitis and gingivitis.

Skin Prick Test with Lipid A was performed on three groups ofindividuals:

Patients with severe marginal periodontitis which does not respond totreatment (refractory patients).

Patients with severe marginal periodontitis which responds to treatment.

Healthy control individuals without marginal periodontitis.

(The test was performed according to a routine procedure as described inAas K, Backman A, Berlin L, Weeke B. Standardization of allergenextracts with appropriate methods. The combined use of skin pricktesting and standardization of allergen extracts. Acta Allergol 1978;33:238-240, and in Malling H-J. The skin prick testing and the use ofhistamine references. Allergy 1984; 39:596-601).

Patient and disease characteristics for the three groups can be found inTable 1 below. GI (gingival index) was used as measure of activedisease. Refractory patients all had a GI>20. The test substance wasLipid A dissolved in water (0.1 mg/ml and 1 mg/ml). Histamine (10 mg/mlin water) and water served as positive and negative controls,respectively. The result was registered after 18 to 24 hours.

TABLE 1 Patient and disease characteristics for the two test groups andthe control group. No significant differences were found between thegroups with marginal periodontitis with respect to gender, age, pocketdepth and number of teeth.

Mean periodontal % female Mean No. of Marginal pocket Mean gingivalTest/control group n % male age teeth bone loss depth index Patientswith severe 14 42.9 45.7 21.1 32.8% 3.2 29.2 marginal periodontitis 57.1which does not respond to treatment (GI > 20) Patients with severe 3844.7 45.4 21.3 30.3% 2.9 11.1 marginal periodontitis 55.3 which respondsto treat- ment (GI ≦ 20) Healthy control indivi- 16 81.2 42.1 — 1.8 mm —— duals without marginal 18.8 periodontitis

All individuals reacted positively to histamine and negatively to thevehicle (water). Students t-test was employed to test levels ofsignificance for differences between test and control groups.

Results from the Skin Prick Test can be found in Table 2 below.

TABLE 2 Skin Prick Test with Lipid A performed on three groups ofindividuals: patients with severe marginal periodontitis which does notrespond to treatment (refractory patients), patients with severemarginal periodontitis which responds to treatment and healthy controlindividuals without marginal periodontitis.

% + with % + with 1 mg 0.1 mg Test/control group n Lipid A P Lipid A PPatients with severe 14 7.1 0.0007↓ 7.1 ns↓↓ marginal periodontitis ↓ns↓ which does not respond 0.0093↓ to treatment (GI > 20) ↓ Patientswith severe 38 57.9 ns↓ 21.1 ns↓ marginal periodontitis which respondsto treat- ment (GI ≦ 20) Healthy control indivi- 16 50.0 18.8 dualswithout marginal periodontitis

Patients with severe marginal periodontitis not responding to treatment(refractory patients) showed almost no positive response to Lipid Awhich was significantly different from the results from patients withsevere marginal periodontitis which responds to treatment and healthycontrol individuals without marginal periodontitis. Refractory patientsalso required higher concentrations of Lipid A to respond. Although theinvention is not restricted to any particular theory, this can beinterpreted as an impaired inflammatory reactivity to periodontitispathogens in refractory patients and the test can be used to screenindividuals to identify those susceptible to severe refractoryperiodontitis before periodontitis has progressed too far. Thus,preventive measures could be specifically directed towards thesepatients.

What is claimed is:
 1. A method for determination of predisposition ofan individual to develop marginal periodontitis comprising: a)performing a screening test on said individual with a first reagentcomprising a surface structure common to anaerobic Gram negativepathogens to elicit a reaction in the individual, wherein the firstreagent is capable of triggering an inflammatory response associatedwith periodontitis and gingivitis in the individual; and b) evaluatingthe reaction, lack of reaction or minor reaction of the individual tothe first reagent of the screening test, indicating the predispositionof the individual to develop marginal periodontitis; wherein step a) isaccompanied by challenge with a negative control to confirm negativereaction; wherein said first reagent comprises a structure selected froma non-carbohydrate entity of surface lipo- or proteoconjugates of saidpathogens, wherein a second reagent is an agonist capable of producingan inflammatory response on challenge of said individual to confirmpositive reaction selected from histamine, serotonin, prostaglandins,leukotrienes and bradykinin, and wherein said negative control ispyrogene free water; and wherein said first reagent comprises Lipid A ina concentration within the range about 0.05 to about 2 mg/ml.